Preparation method and application of near-infrared two-region linear fluorescent sensor

Abstract

The application provides a preparation method and application of a near-infrared two-region fluorescent sensor, and chiral single-walled carbon nanotubes are separated by using a double water phase extraction method; materials in the separation process include deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate and cholic acid sodium hydrate; the chiral single-walled carbon nanotubes are dispersed and mixed with 4-nitrobenzene tetrafluoroboric acid diazonium salt to obtain the fluorescent sensor; high-purity (6,5)-SWCNT is efficiently and simply separated by using the double water phase extraction technology, the prepared SWCNT-NBD nanomaterial has good optical performance, including bright light emission in the near-infrared two-region, has good penetration capacity on biological tissues, the fluorescent detector (SWCNT-NBD) has low detection line, wide linear range, good selectivity on DA, and is convenient to prepare and low in cost.

Description

Technical Field

[0001] This invention relates to the field of nanosensor technology, and in particular to a method for preparing a near-infrared II fluorescence sensor and its application. Background Technology

[0002] Dopamine (DA), a catecholamine neurotransmitter, plays a crucial role in human health. Clinical studies have shown that patients with depression experience impaired DA perception, leading to a loss of pleasure—that is, a disruption in their ability to experience happiness. Furthermore, research indicates that other mental illnesses are also related to DA. In the central nervous system, DA levels are also associated with diseases such as Parkinson's disease and Alzheimer's disease. In living organisms, DA concentrations below 100 nM are significant. For example, the health of the human eye can be directly assessed by DA levels. Martin et al., using high-performance liquid chromatography-electrochemical methods, found that the average DA concentration in the tears of healthy individuals was 58 ± 33 nM, while the DA concentration in the tears of people with myopia was lower. Therefore, developing DA fluorescent probes with high sensitivity, low detection limits, and outstanding selectivity is of great importance for the detection, prevention, diagnosis, and treatment of diseases, but it also presents a significant challenge.

[0003] Currently, detection techniques for dopamine (DA) include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), electrochemical methods, spectroscopic methods, and colorimetric methods. However, these techniques are all less than satisfactory in terms of sample preparation, detection stability, response time, interference resistance, and detection cost. Cyclic voltammetry (CV) in electrochemical methods has become a commonly used method for DA detection due to its simplicity, fast response, and low cost. However, in untreated human body fluid samples, DA is often found alongside molecules with similar structures, such as adrenaline (EP), which belongs to the catecholamine neurotransmitter class and has similar redox potentials. This poses a challenge to DA detection. Furthermore, redox reactions occurring at the electrode can contaminate the electrode, shortening its lifespan, resulting in low detection stability and poor performance. Electrochemical methods are also easily affected by environmental factors such as pH and temperature. Fluorescent sensors have excellent potential in bioimaging; however, they still suffer from complex preparation processes, limitations in spatiotemporal resolution, selectivity, and stability. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing a near-infrared II fluorescence sensor. This invention solves the problems of high detection cost, poor selectivity, low sensitivity, and inability to achieve linear sensing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] A method for preparing a near-infrared II linear fluorescence sensor involves separating chiral single-walled carbon nanotubes using an aqueous two-phase extraction method. The materials used in the separation process include deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate, and sodium cholate hydrate. The chiral single-walled carbon nanotubes are dispersed and mixed with 4-nitrobenzene tetrafluoroborate diazonium salt to obtain the fluorescence sensor.

[0007] In this invention, chiral single-walled carbon nanotube powder is dispersed in a 1% sodium deoxycholate solution at a ratio of 1 mg / mL to obtain a first mixed solution; the first mixed solution is subjected to high-precision ultrasonication and centrifugation, and the upper 80% clear liquid is collected to obtain a second mixed solution; 2 mass% dextran, 12.5 mass% polyethylene glycol, 0.5 g / L sodium deoxycholate, and 18 g / L sodium dodecyl sulfate are mixed to obtain a first separated solution;

[0008] Take the second mixed solution, deionized water, 20 wt / v% dextran solution and 40 wt / v% polyethylene glycol solution in a volume ratio of 1:3:4:2 and place them in centrifuge tubes to obtain the third mixed solution;

[0009] The third mixed solution was centrifuged at 8000 rpm for 1 minute, and the third mixed solution separated into upper and lower phases. The lower phase was taken as the fourth mixed solution.

[0010] Add the fourth mixed solution and an equal volume of the first separated solution to a centrifuge tube and mix well. Centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases. Take the lower phase as the fifth mixed solution.

[0011] Add the fifth mixed solution and an equal volume of the first separated solution to a centrifuge tube, mix well, and centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases, and the lower phase will be taken as the sixth mixed solution.

[0012] Add the sixth mixed solution and an equal volume of the first separated solution to a centrifuge tube, mix well, and centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases, and the lower phase will be taken as the seventh mixed solution.

[0013] Add the seventh mixed solution and an equal volume of the first separated solution to a centrifuge tube and mix well. Centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture is divided into two phases, the upper phase of which is a high-purity (6,5)-chiral single-walled carbon nanotube dispersion.

[0014] The obtained (5,6) chiral single-walled carbon nanotubes were diluted with 0.1 wt.% sodium dodecyl sulfate solution to an absorbance of 0.15; the first detection solution was obtained.

[0015] A 20 nM solution of 4-nitrophenyltetrafluoroborate diazonium salt was added to the first detection solution to obtain a fluorescence sensor.

[0016] An application of a fluorescence sensor, prepared using the method of the near-infrared II linear fluorescence sensor of the present invention, is used for linear detection of dopamine.

[0017] The present invention adopts the above technical solution, and compared with the prior art, the following technical effects are achieved:

[0018] This invention provides a method for preparing a near-infrared II fluorescence sensor and its application. The invention efficiently and easily separates high-purity (6,5)-SWCNT using a two-phase aqueous extraction technique. The prepared SWCNT-NBD nanomaterial has good optical properties, including bright near-infrared II emission and good penetration into biological tissues. The fluorescence detector (SWCNT-NBD) has a low detection line, a wide linear range, good selectivity for DA, and is convenient and low in cost to prepare. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A schematic diagram of the detection principle of a fluorescence sensor for dopamine detection provided in an embodiment of the present invention;

[0021] Figure 2 The steady-state fluorescence spectrum, ultraviolet-visible-near-infrared absorption spectrum, and Raman spectrum of the first detection solution before and after the addition of dopamine are provided in the embodiments of the present invention.

[0022] Figure 3 The linear detection effect diagram provided in the embodiment of the present invention;

[0023] Figure 4 This is a selective detection result diagram provided in an embodiment of the present invention;

[0024] Figure 5 The image shows the detection results of actual DA concentration in a physiological saline environment provided in this embodiment of the invention. Detailed Implementation

[0025] A method for preparing a near-infrared II linear fluorescence sensor involves separating chiral single-walled carbon nanotubes (SWCNTs) using an aqueous two-phase extraction method. The materials used in the separation process include deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate, and sodium cholate hydrate. The chiral SWCNTs are dispersed and mixed with diazonium salt of 4-nitrophenyltetrafluoroborate (NBD) to obtain the fluorescence sensor.

[0026] A method for preparing a near-infrared II linear fluorescence sensor involves dispersing SWCNT powder at a ratio of 1 mg / mL in a 1% sodium deoxycholate solution to obtain a first mixed solution; after subjecting the first mixed solution to ultrasonication and centrifugation, collecting the upper 80% clear liquid to obtain a second mixed solution; and mixing 2 mass% dextran, 12.5 mass% polyethylene glycol, 0.5 g / L sodium deoxycholate, and 18 g / L sodium dodecyl sulfate to obtain a first separation solution.

[0027] Take the second mixed solution, deionized water, 20 wt / v% dextran solution and 40 wt / v% polyethylene glycol solution in a volume ratio of 1:3:4:2 and place them in centrifuge tubes to obtain the third mixed solution;

[0028] The third mixed solution was centrifuged at 8000 rpm for 1 minute, and the third mixed solution separated into upper and lower phases. The lower phase was taken as the fourth mixed solution.

[0029] Add the fourth mixed solution and an equal volume of the first separated solution to a centrifuge tube and mix well. Centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases. Take the lower phase as the fifth mixed solution.

[0030] Add the fifth mixed solution and an equal volume of the first separated solution to a centrifuge tube, mix well, and centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases, and the lower phase will be taken as the sixth mixed solution.

[0031] Add the sixth mixed solution and an equal volume of the first separated solution to a centrifuge tube, mix well, and centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases, and the lower phase will be taken as the seventh mixed solution.

[0032] Add the seventh mixed solution and an equal volume of the first separated solution to a centrifuge tube and mix well. Centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture is divided into two phases, the upper phase of which is the high-purity (6,5)-SWCNT dispersion.

[0033] The obtained (6,5)-SWCNT was diluted with 0.1 wt.% sodium dodecyl sulfate solution to an absorbance of 0.15; the first detection solution was obtained.

[0034] A 20 nM NBD solution was added to the first detection solution to obtain a fluorescence sensor.

[0035] Preferably, the stirring speed of the first separation preparation solution of the present invention is 1200 rpm.

[0036] Preferably, the stirring time for the first mixed solution of the present invention is 20 min; the tip ultrasound of the first mixed solution of the present invention is performed under ice bath conditions; the tip diameter for the tip ultrasound of the first mixed solution of the present invention is 6 mm; the power for the tip ultrasound of the first mixed solution of the present invention is 1200 rpm; the tip ultrasound time for the first mixed solution of the present invention is 60 min; the centrifugation speed for the first mixed solution of the present invention is 12000 rpm; and the centrifugation time for the first mixed solution of the present invention is 10 min.

[0037] Preferably, when mixing the third mixed solution of the present invention, the speed of the vortex mixer is 2800 rpm; and the mixing time is 1 min.

[0038] Preferably, when mixing the various mixed solutions of the present invention, the speed of the vortex mixer is 2800 rpm; and the mixing time is 1 min.

[0039] Preferably, the absorbance of the (6,5)-SWCNT dispersion of the present invention is measured under ultraviolet-visible-near-infrared absorption spectroscopy.

[0040] Preferably, the NBD solution of the present invention is added during the stirring of the first detection solution.

[0041] Preferably, the stirring speed of the first detection solution of the present invention is 800 rpm.

[0042] An application of a fluorescence sensor, prepared using the method of the near-infrared II linear fluorescence sensor of the present invention, is used for linear detection of dopamine.

[0043] The beneficial effects of the present invention are verified using the following embodiments:

[0044] Example 1: The preparation method of the fluorescence sensor for linear dopamine detection in this example is carried out according to the following steps:

[0045] 1. Add 20 mg of single-walled carbon nanotube powder to 20 mL of sodium deoxycholate solution with a concentration of 1% wt / v to obtain the first mixed solution; add 1 g of dextran, 6.25 g of polyethylene glycol, 0.035 g of sodium deoxycholate, and 0.9 g of sodium dodecyl sulfate to 50 mL of deionized water and mix to obtain the first separated solution.

[0046] 2. The first mixed solution is subjected to tip sonication in an ice bath to uniformly disperse the carbon nanotube powder. Then, the undispersed carbon nanotube bundles and impurities are removed by centrifugation. The upper 80% of the clear liquid after centrifugation is taken to obtain the second mixed solution.

[0047] 3. Add 6 mL of deionized water, 2 mL of the second mixed solution, 8 mL of 20% dextran solution and 4 mL of 40% polyethylene glycol solution to a 50 mL centrifuge tube, mix well to obtain the third mixed solution.

[0048] 4. Centrifuge the third mixed solution. The lower phase after centrifugation is the fourth mixed solution.

[0049] 5. Take out the fourth mixed solution and add it to a 50mL centrifuge tube. Add an equal volume of the first separation solution and mix well to obtain the fifth mixed solution.

[0050] 6. Repeat step 5 three times to obtain a (6,5)-SWCNT dispersion from the superphase after centrifugation.

[0051] 7. Dilute the (6,5)-SWCNT dispersion with 0.1 wt / v% sodium dodecyl sulfate solution, and dilute the (6,5)-SWCNT dispersion to an absorbance of 0.15 under the characterization of UV-Vis-NIR absorption spectrometry.

[0052] 8. Take 2 mL of the above (6,5)-SWCNT dispersion with an optical density of 0.15 and mix it with 20 μL of NBD solution with a concentration of 1 nM to obtain the first dopamine probe solution;

[0053] 9. Weigh 0.9 mg of dopamine and add it to a 10 mL volumetric flask. Make up to volume with deionized water, and sonicate until the dopamine is completely dissolved. Then dilute it 1000 times with deionized water to obtain a dopamine stock solution with a concentration of 1.96 μM.

[0054] 10. Weigh five different biomass small molecules (adrenaline, serotonin, glutathione, uric acid and gallic acid) and add them to 10 mL volumetric flasks respectively. Use deionized water and sonicate to mix evenly, so that the different kinds of biomass small molecules are completely dissolved, and obtain stock solutions of different kinds of biomass small molecules with a concentration of 1.96 μM.

[0055] The fluorescent probe for linear dopamine detection prepared in this embodiment was characterized by steady-state fluorescence spectroscopy, UV-Vis-NIR absorption spectroscopy, and Raman spectroscopy. The detection results are as follows: Figure 2 As shown. From Figure 2As can be seen from the fluorescence spectrum, in addition to the intrinsic fluorescence emission of (6,5)-SWCNT at 980 nm, a red-shifted and stronger characteristic absorption peak was generated after the addition of dopamine, indicating that the fluorescent probe for linear detection of dopamine was successfully prepared.

[0056] This implementation also includes performance testing of the dopamine fluorescence sensor:

[0057] 1. Add 1, 10, 20, 40, 60, 60, 80, and 100 μL of 1.96 μM dopamine stock solution and 100 mL of different types of 1.96 μM biomass small molecule stock solutions to 2 mL of the first dopamine probe solution to obtain mixed solutions. During fluorescence detection, magnetic stirring at 800 rpm is performed. After adding the target analyte, allow the system to stabilize for 10 min before starting fluorescence detection.

[0058] II. Using 565 nm as the excitation wavelength and the wavelength range of 800–1400 nm as the detection interval, the fluorescence emission spectra of (6,5)-SWCNT dispersion, the first dopamine probe solution, dopamine stock solution, and mixtures of different types of biomass small molecule stock solutions with the first dopamine probe solution were detected. The detection results are as follows: Figure 2 , Figure 3 As shown, the linear detection results for dopamine are as follows: Figure 4 As shown.

[0059] like Figure 1 As shown, the dopamine fluorescence sensor of the present invention utilizes the reducing property of dopamine (DA) to reduce 4-nitrobenzene tetrafluoroborate diazonium salt (NBD) to aryl radicals during detection, which are then covalently attached to the surface of carbon nanotubes, thereby destroying the SP2 lattice structure of the carbon nanotubes and forming a quantum potential well, resulting in bright luminescence in the near-infrared II region.

[0060] Figure 2 In the graph 'a', with emission wavelength on the horizontal axis, optical density on the left axis, and fluorescence intensity on the right axis, it can be seen that the near-infrared fluorescence emission peak of the first dopamine probe solution under 565nm excitation light is located at 980nm. However, after the addition of dopamine, a redshifted fluorescence peak with stronger emission intensity is generated at 1140nm. Figure 2 The two-dimensional fluorescence contour plot in section b, drawn with the excitation wavelength as the vertical axis and the emission wavelength as the horizontal axis, illustrates this point more intuitively. Figure 2 The Raman spectrum of carbon nanotubes (C) was plotted with Raman intensity as the ordinate and wavenumber as the abscissa. The significant increase in the D / G ratio after the addition of DA indicates that the SP of carbon nanotubes... 2 The crystal lattice was destroyed, forming SP. 3The quantum defects have prevented the verification of fluorescence emission at 1140 nm.

[0061] Combination Figure 3 In Figure 'a', a three-dimensional waterfall plot was drawn with dopamine concentration, fluorescence intensity, and emission wavelength as coordinate axes, visually demonstrating the relationship between different dopamine concentrations and the luminescence intensity of the fluorescent probe. It can be clearly seen that the intensity of the characteristic fluorescence peak at 1140 nm increases with increasing dopamine concentration. Combined with... Figure 3 In Figure b, the average intensity (I-I0) / I0 of the fluorescence characteristic peak at 1140 nm before and after dopamine addition is plotted on the ordinate, and the dopamine concentration on the abscissa. Analysis revealed a linear relationship between the fluorescence peak intensity and dopamine concentration. A linear function was fitted between the fluorescence emission intensity (y) and dopamine concentration (x): y = 0.03x + 0.53, with a coefficient of determination R0. 2 Up to 0.999.

[0062] Figure 4 In the graph, other small biomolecules are plotted on the x-axis, and the average intensity (I-I0) / I0 of the fluorescence characteristic peak at 1140 nm before and after the addition of other small biomolecules is plotted on the y-axis. The comparison shows that almost no red-shifted fluorescence peaks appear after the addition of other small biomolecule solutions, demonstrating good selectivity. Therefore, it can be seen that the first dopamine probe solution prepared in this embodiment has the ability to simultaneously perform linear detection and selective recognition of dopamine.

[0063] III. When using the probe solution A prepared in this embodiment for dopamine detection, in order to evaluate its actual detection capability, physiological saline was used to simulate the human body fluid environment for dopamine detection. The specific method is as follows: the (6,5)-SWCNT dispersion was diluted with physiological saline solution, and the (6,5)-SWCNT dispersion was diluted to an absorbance of 0.15 under the characterization of ultraviolet-visible-near-infrared absorption spectroscopy to obtain the second dopamine probe solution;

[0064] Add 50 μL of 1.96 μM dopamine stock solution to 2 mL of the second dopamine probe solution to achieve a dopamine concentration of 50 nM in the final test system. After thorough mixing, immediately perform fluorescence testing. The actual detection performance results are as follows: Figure 5 As shown in Figure a, fluorescence intensity is the ordinate and dopamine concentration is the abscissa. Figure 5 As shown in b, Figure 5 Substitute the test results of Chinese a into Figure 3The linear conclusion in section b was verified (using the average intensity of the fluorescence characteristic peak at 1140 nm before and after dopamine addition, (I-I0) / I0, as the ordinate and dopamine concentration as the abscissa). Analysis revealed that the final detection results conformed to the derived function of fluorescence intensity versus dopamine concentration. This indicates that even in a liquid-phase environment with physiological saline, the dopamine fluorescence sensor can still accurately detect dopamine concentration. This demonstrates that the linear dopamine detection fluorescence sensor of this embodiment has the potential for practical application.

Claims

1. A method for fabricating a near-infrared II linear fluorescence sensor, characterized in that: Chiral single-walled carbon nanotubes were separated using an aqueous two-phase extraction method. The materials used in the separation process included deionized water, polyethylene glycol, dextran, sodium dodecyl sulfate, sodium deoxycholate, and sodium cholate hydrate. The chiral single-walled carbon nanotubes were dispersed and mixed with 4-nitrobenzene tetrafluoroborate diazonium salt to obtain a fluorescent sensor. Chiral single-walled carbon nanotube powder was dispersed in a 1% sodium deoxycholate solution at a ratio of 1 mg / mL to obtain a first mixed solution; the first mixed solution was subjected to high-precision ultrasonication and centrifugation, and 80% of the supernatant was collected to obtain a second mixed solution. The first separation solution was prepared by mixing 2 mass% dextran, 12.5 mass% polyethylene glycol, 0.5 g / L sodium deoxycholate, and 18 g / L sodium dodecyl sulfate. Take the second mixed solution, deionized water, 20 wt / v% dextran solution and 40 wt / v% polyethylene glycol solution in a volume ratio of 1:3:4:2 and place them in centrifuge tubes to obtain the third mixed solution; The third mixed solution was centrifuged at 8000 rpm for 1 minute, and the third mixed solution separated into upper and lower phases. The lower phase was taken as the fourth mixed solution. Add the fourth mixed solution and an equal volume of the first separated solution to a centrifuge tube and mix well. Centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases. Take the lower phase as the fifth mixed solution. Add the fifth mixed solution and an equal volume of the first separated solution to a centrifuge tube, mix well, and centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases, and the lower phase will be taken as the sixth mixed solution. Add the sixth mixed solution and an equal volume of the first separated solution to a centrifuge tube, mix well, and centrifuge at 8000 rpm for 1 minute. After centrifugation, the mixture will separate into two phases, and the lower phase will be taken as the seventh mixed solution. The seventh mixed solution and an equal volume of the first separated solution were added to a centrifuge tube and mixed thoroughly. The mixture was then centrifuged at 8000 rpm for 1 minute. After centrifugation, the mixture separated into two phases, the upper phase of which was a high-purity (6,5)-chiral single-walled carbon nanotube dispersion. The obtained (6,5)-chiral single-walled carbon nanotubes were diluted with 0.1 wt.% sodium dodecyl sulfate solution to an absorbance of 0.15 to obtain the first detection solution. A 20 nM solution of 4-nitrobenzenetetrafluoroborate diazonium salt was added to the first detection solution to obtain a fluorescence sensor.

2. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: The first separation preparation solution was stirred at a speed of 1200 rpm.

3. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: The stirring time for the first mixed solution is 20 min; the tip sonication of the first mixed solution is performed under ice bath conditions; the tip diameter for the tip sonication of the first mixed solution is 6 mm; the power for the tip sonication of the first mixed solution is 1200 rpm; the tip sonication time for the first mixed solution is 60 min; the centrifugation speed for the first mixed solution is 12000 rpm; and the centrifugation time for the first mixed solution is 10 min.

4. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: When mixing the third mixed solution, the speed of the vortex mixer is 2800 rpm; the mixing time is 1 min.

5. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: When mixing the above mixed solutions, the speed of the vortex mixer is 2800 rpm; the mixing time is 1 min.

6. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: The absorbance of the (6,5)-chiral single-walled carbon nanotube dispersion was determined by UV-Vis-NIR absorption spectroscopy.

7. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: The 4-nitrobenzenetetrafluoroborate diazonium salt solution was added while the first detection solution was being stirred.

8. The method for fabricating a near-infrared II linear fluorescence sensor according to claim 1, characterized in that: The stirring speed for the first test solution was 800 rpm.

9. An application of a fluorescence sensor, prepared using the method of the near-infrared two-region linear fluorescence sensor according to any one of claims 1-8, characterized in that... Used for linear detection of dopamine.

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